The urothelium maintains an active set of defenses against bacterial infection. These include static defenses in the form of surface molecules that inhibit bacterial attachment and active responses to eliminate an established bacterial infection. The long-term aim of this study is to elucidate the mechanisms by which antibacterial defenses are modulated by infection. Our hypothesis is that in an environment of increasing bacterial resistance to antibiotics, understanding the natural mechanisms of defense should permit their augmentation as a means to prevent or treat infection, particularly in vulnerable populations. Enterococcus spp. rank second among the leading cause of bacterial UTIs. The high antibiotic resistance prevalent among this genus makes treatment of enterococcal infections a therapeutic challenge. Previous research in our laboratory has established models for urothelium grown in 3 dimensions that closely mimics both the morphology and functional genomics of the bladder urothelium. In this model system, the environment of the urothelial cells can be controlled precisely and specific modulatory proteins can be added, or the actions of specific genes can be inhibited or augmented in a realistic cell culture environment. We will ask two broad questions: 1. How does the urothelium respond to bacterial infection? 2. Can altering expression of specific genes in the urothelium modulate the response to bacterial infection? This approach will integrate closely with independent bacterial and animal model studies carried out by other members of this collaborative research group. Our model permits mechanistic investigations in which the role of specific molecules in the response of urothelium to infection can be tested. Our main objective for this period of support is to expand on current cancer-related cDNA array technology in our laboratory to investigate genome-wide changes in the expression of human urothelial cells in 3-dimensional culture as they experience infection with enterococcus. We plan to use the Clontech system on plastic consisting of over 8,000 named human genes. The aims are to cluster genes according to their behavior over time, identify the key signaling and response pathways that are involved in the response of urothelial cells to infection, and learn how to manipulate and interpret large-scale genomic data. These data will form an essential element in either an R01 or a program project and will permit the PI to develop a new line of urologic research.